MicroRNAs and uses thereof

ABSTRACT

Described herein are novel polynucleotides associated with prostate and lung cancer. The polynucleotides are miRNAs and miRNA precursors. Related methods and compositions that can be used for diagnosis, prognosis, and treatment of those medical conditions are disclosed. Also described herein are methods that can be used to identify modulators of prostate and lung cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 11/130,645,filed May 16, 2005, which is a continuation of International ApplicationNo. PCT/US2005/16986, filed May 14, 2005, which is acontinuation-in-part of U.S. application Ser. No. 10/709,572 filed May14, 2004, and U.S. application Ser. No. 10/709,577, filed May 14, 2004,and which claims the benefit of U.S. Provisional Application No.60/666,340, filed Mar. 30, 2005, U.S. Provisional Application No.60/665,094, filed Mar. 25, 2005, U.S. Provisional Application No.60/662,742, filed Mar. 17, 2005, U.S. Provisional Application No.60/593,329, filed Jan. 6, 2005, U.S. Provisional Application No.60/593,081, filed Dec. 8, 2004, U.S. Provisional Application No.60/522,860, filed Nov. 15, 2004, U.S. Provisional Application No.60/522,457, filed Oct. 4, 2004, U.S. Provisional Application No.60/522,452, filed Oct. 3, 2004, and U.S. Provisional Application No.60/522,449, filed Oct. 3, 2004, the contents of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates in general to microRNA molecules as well asvarious nucleic acid molecules relating thereto or derived therefrom.

REFERENCES TO THE SEQUENCE LISTING AND COMPUTER PROGRAM LISTINGAPPENDICES

Applicant hereby makes reference to the sequence listing and computerprogram listing appendices that were submitted on compact disc. Thesequence listing consists of a filed named “SeqList.txt,” (168,151bytes), created on Mar. 5, 2010. The computer program listing appendixconsists of the following files: “Table 1.bat” (968 KB), “Table 2.bat”(1327 KB), “Table 3.bat” (9 KB), “Table 4.bat” (10,857 KB), “Table5.bat” (986 KB), “Table 6.bat” (38 KB), “Table 7.bat” (2 KB), “Table8.bat” (4 KB), “Table 9.bat” (190 KB), “Table 10_(—)001.bat” (716,801KB), “Table 10_(—)002.bat” (716,801 KB), “Table 10_(—)003.bat” (105,669KB), “Table 11A.bat” (634,769 KB), “Table 11B.bat” (634,770 KB), “Table11C.bat” (113,878 KB), “Table 12.bat” (585,921 KB), “Table 13A.bat”(634,767 KB), “Table 13B.bat” (51,584 KB), “Table 14.bat” (75 KB),“Table 15.bat” (68 KB), “Table 16.bat” (774 KB), and “Table 17.bat”(3183 KB), all of which were created on Feb. 15, 2010. The sequencelisting and computer program listing appendices are all incorporatedherein by reference.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are short RNA oligonucleotides of approximately 22nucleotides that are involved in gene regulation. MicroRNAs regulategene expression by targeting mRNAs for cleavage or translationalrepression. Although miRNAs are present in a wide range of speciesincluding C. elegans, Drosophilla and humans, they have only recentlybeen identified. More importantly, the role of miRNAs in the developmentand progression of disease has only recently become appreciated.

As a result of their small size, miRNAs have been difficult to identifyusing standard methodologies. A limited number of miRNAs have beenidentified by extracting large quantities of RNA. MiRNAs have also beenidentified that contribute to the presentation of visibly discernablephenotypes. Expression array data shows that miRNAs are expressed indifferent developmental stages or in different tissues. The restrictionof miRNAs to certain tissues or at limited developmental stagesindicates that the miRNAs identified to date are likely only a smallfraction of the total miRNAs.

Computational approaches have recently been developed to identify theremainder of miRNAs in the genome. Tools such as MiRscan and MiRseekerhave identified miRNAs that were later experimentally confirmed. Basedon these computational tools, it has been estimated that the humangenome contains 200-255 miRNA genes. These estimates are based on anassumption, however, that the miRNAs remaining to be identified willhave the same properties as those miRNAs already identified. Based onthe fundamental importance of miRNAs in mammalian biology and disease,the art needs to identify unknown miRNAs. The present inventionsatisfies this need and provides a significant number of miRNAs and usestherefore.

SUMMARY OF THE INVENTION

The present invention is related to an isolated nucleic acid comprisinga sequence of a pri-miRNA, pre-miRNA, miRNA, miRNA*, anti-miRNA, or amiRNA binding site, or a variant thereof. The nucleic acid may comprisethe sequence of a hairpin referred to in Table 1; the sequence ofsequence identifiers 6757248-6894882 of the Sequence Listing of U.S.patent application Ser. No. 10/709,572, which is incorporated herein byreference; the sequence of sequence identifiers 1-6318 or 18728-18960 ofthe Sequence Listing of U.S. Provisional Patent Application No.60/655,094, which is incorporated herein by reference; the sequence of amiRNA referred to in Table 1; the sequence of sequence identifiers1-117750 or 6894883-10068177 of the Sequence Listing of U.S. patentapplication Ser. No. 10/709,572, which is incorporated herein byreference; the sequence of sequence identifiers 6319-18727 or18961-19401 of the Sequence Listing of U.S. Provisional PatentApplication No. 60/655,094, which is incorporated herein by reference;the sequence of a target gene binding site referred to in Table 4; thesequence of sequence identifiers 117751-6757247 of the Sequence Listingof U.S. patent application Ser. No. 10/709,572, which is incorporatedherein by reference; a complement thereof; or a sequence comprising atleast 12 contiguous nucleotides at least 60% identical thereto. Theisolated nucleic acid may be from 5-250 nucleotides in length.

The present invention is also related to a probe comprising the nucleicacid. The probe may comprise at least 8-22 contiguous nucleotidescomplementary to a miRNA referred to in Table 2 as differentiallyexpressed in prostate cancer or lung cancer.

The present invention is also related to a plurality of the probes. Theplurality of probes may comprise at least one probe complementary toeach miRNA referred to in Table 2 as differentially expressed inprostate cancer. The plurality of probes may also comprise at least oneprobe complementary to each miRNA referred to in Table 2 asdifferentially expressed in lung cancer.

The present invention is also related to a composition comprising aprobe or plurality of probes.

The present invention is also related to a biochip comprising a solidsubstrate, said substrate comprising a plurality of probes. Each of theprobes may be attached to the substrate at a spatially defined address.The biochip may comprise probes that are complementary to a miRNAreferred to in Table 2 as differentially expressed in prostate cancer.The biochip may also comprise probes that are complementary to a miRNAreferred to in Table 2 as differentially expressed in lung cancer.

The present invention is also related to a method of detectingdifferential expression of a disease-associated miRNA. A biologicalsample may be provide and the level of a nucleic acid measured that isat least 70% identical to a sequence of a miRNA referred to in Table 1;the sequence of sequence identifiers 1-117750 or 6894883-10068177 of theSequence Listing of U.S. patent application Ser. No. 10/709,572, whichis incorporated herein by reference; the sequence of sequenceidentifiers 6319-18727 or 18961-19401 of the Sequence Listing of U.S.Provisional Patent Application No. 60/655,094, which is incorporatedherein by reference; or variants thereof. A difference in the level ofthe nucleic acid compared to a control is indicative of differentialexpression.

The present invention is also related to a method of identifying acompound that modulates a pathological condition. A cell may be providedthat is capable of expressing a nucleic acid at least 70% identical to asequence of a miRNA referred to in Table 1; the sequence of sequenceidentifiers 1-117750 or 6894883-10068177 of the Sequence Listing of U.S.patent application Ser. No. 10/709,572, which is incorporated herein byreference; the sequence of sequence identifiers 6319-18727 or18961-19401 of the Sequence Listing of U.S. Provisional PatentApplication No. 60/655,094, which is incorporated herein by reference;or variants thereof. The cell may be contacted with a candidatemodulator and then measuring the level of expression of the nucleicacid. A difference in the level of the nucleic acid compared to acontrol identifies the compound as a modulator of a pathologicalcondition associated with the nucleic acid.

The present invention is also related to a method of inhibitingexpression of a target gene in a cell. Into the cell, a nucleic acid maybe introduced in an amount sufficient to inhibit expression of thetarget gene. The target gene may comprise a binding site substantiallyidentical to a binding site referred to in Table 4; a sequence ofsequence identifiers 117751-6757247 of the Sequence Listing of U.S.patent application Ser. No. 10/709,572, which is incorporated herein byreference; or a variant thereof. The nucleic acid may comprise asequence of SEQ ID NOS: 1-760616; a sequence of sequence identifiers1-117750 and 6757248-10068177 of the Sequence Listing of U.S. patentapplication Ser. No. 10/709,572, which is incorporated herein byreference; a sequence set forth on the Sequence Listing of U.S.Provisional Patent Application No. 60/655,094, which is incorporatedherein by reference; or a variant thereof. Expression of the target genemay be inhibited in vitro or in vivo.

The present invention is also related to a method of increasingexpression of a target gene in a cell. Into the cell, a nucleic acid maybe introduced in an amount sufficient to inhibit expression of thetarget gene. The target gene may comprise a binding site substantiallyidentical to a binding site referred to in Table 4; a sequence ofsequence identifiers 117751-6757247 of the Sequence Listing of U.S.patent application Ser. No. 10/709,572, which is incorporated herein byreference; or a variant thereof. The nucleic acid may comprise asequence substantially complementary to SEQ ID NOS: 1-760616; a sequenceof sequence identifiers 1-117750 and 6757248-10068177 of the SequenceListing of U.S. patent application Ser. No. 10/709,572, which isincorporated herein by reference; a sequence set forth on the SequenceListing of U.S. Provisional Patent Application No. 60/655,094, which isincorporated herein by reference; or a variant thereof. Expression ofthe target gene may be inhibited in vitro or in vivo. Expression of thetarget gene may be increased in vitro or in vivo.

The present invention is also related to a method of treating a patientwith a disorder set forth on Table 6 comprising administering to apatient in need thereof a nucleic acid comprising a sequence of SEQ IDNOS: 1-760616; a sequence set forth on the Sequence Listing of U.S.patent application Ser. No. 10/709,572, which is incorporated herein byreference; a sequence set forth on the Sequence Listing of U.S.Provisional Patent Application No. 60/655,094, which is incorporatedherein by reference; or a variant thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates a model of maturation for miRNAs.

FIG. 2 shows a schematic illustration of the MC19cluster on 19q13.42.Panel A shows the ˜500,000 bp region of chromosome 19, from 58,580,001to 59,080,000 (according to the May 2004 USCS assembly), in which thecluster is located including the neighboring protein-coding genes. TheMC19-1 cluster is indicated by a rectangle. Mir-371, mir-372, andmir-373 are indicted by lines. Protein coding genes flanking the clusterare represented by large arrow-heads. Panel B shows a detailed structureof the MC19-1 miRNA cluster. A region of ˜102,000 bp, from 58,860,001 to58,962,000 (according to the May 2004 USCS assembly), is presented.MiRNA precursors are represented by a black bars. It should be notedthat all miRNAs are at the same orientation from left to right. Shadedareas around miRNA precursors represent repeating units in which theprecursor is embedded. The location of mir-371, mir-372, and mir-373, isalso presented.

FIG. 3 is a graphical representation of multiple sequence alignment of35 human repeat units at distinct size of ˜690 nt (A) and 26 chimpanzeesrepeat units (B). The graph was generated by calculating a similarityscore for each position in the alignment with an averaging slidingwindow of 10 nt (Maximum score—1, minimum score—0). The repeat unitsequences were aligned by ClustalW program. Each position of theresulting alignment was assigned a score which represented the degree ofsimilarity at this position. The region containing the miRNA precursorsis bordered by vertical lines. The exact location of the mature miRNAsderived from the 5′ stems (5p) and 3′ stems (3p) of the precursors isindicted by vertical lines.

FIG. 4 shows sequence alignments of the 43 A-type pre-miRNAs of theMC19-1 cluster. Panel A shows the multiple sequence alignment with thePosition of the mature miRNAs marked by a frame. The consensus sequenceis shown at the bottom. Conserved nucleotides are colored as follows:black-100%, dark grey-80% to 99%, and clear grey-60% to 79%. Panel Bshows alignments of consensus mature A-type miRNAs with the upstreamhuman cluster of mir-371, mir-372, miR-373. Panel C shows alignments ofconsensus mature A-type miRNAs with the hsa-mir-371-373 mouseorthologous cluster. The miRNAs hsa-miR-A1 through hsa-miR-A43 of panelA have SEQ ID NOs: 760625 through 760667, respectively. In panel B, thefollowing miRNAs have the following sequences: hsa-miR-371, 3p (SEQ IDNO: 760668), hsa-miR-372, 3p (SEQ ID NO: 760669), hsa-miR-373, 3p (SEQID NO: 760670), hsa-miR-A-3p (consensus) (SEQ ID NO: 760671),hsa-miR-373-5-p (SEQ ID NO: 760672), hsa-miR-A-5p (consensus) (SEQ IDNO: 760678). In panel C, the following miRNAs have the followingsequences: hsa-miR-302a (SEQ ID NO: 760673), hsa-miR-302b (SEQ ID NO:760674), hsa-miR-302c (SEQ ID NO: 760675), hsa-miR-302d (SEQ ID NO:760676), and hsa-miR-A-3p (consensus) (SEQ ID NO: 760677).

FIG. 5 shows expression analysis of the MC19-1 miRNAs. Panel A shows aNorthern blot analysis of two selected A-type miRNAs. Expression wasanalyzed using total RNA from human brain (B), liver (L), thymus (T),placenta (P) and HeLa cells (H). The expression of mir-98 and ethidiumbromide staining of the tRNA band served as control. Panel B showsRT-PCR analysis of the mRNA transcript containing the A-type miRNAprecursors. Reverse transcription of 5ÿg total RNA from placenta wasperformed using oligo-dT. This was followed by PCR using the denotedprimers (indicated by horizontal arrows). The region examined isillustrated at the top. Vertical black bars represent the pre-miRNA;shaded areas around the pre-miRNAs represent the repeating units; thelocation of four ESTs is indicted at the right side; the poly-A site, asfound in the ESTs and located downstream to a AATAAA consensus, isindicated by a vertical arrow. The fragments expected from RT-PCR usingthree primer combinations are indicated below the illustration of thecluster region. The results of the RT-PCR analysis are presented belowthe expected fragments. Panel C shows the sequencing strategy of the FR2fragment. The fragment was cloned into the pTZ57R\T vector and sequencedusing external and internal primers.

DETAILED DESCRIPTION

The present invention provides nucleotide sequences of miRNAs,precursors thereto, targets thereof and related sequences. Such nucleicacids are useful for diagnostic purposes, and also for modifying targetgene expression. Other aspects of the invention will become apparent tothe skilled artisan by the following description of the invention.

1. Definitions

Before the present compounds, products and compositions and methods aredisclosed and described, it is to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting. It must be noted that, as used inthe specification and the appended claims, the singular forms “a,” “an”and “the” include plural referents unless the context clearly dictatesotherwise.

a. Animal

“Animal” as used herein may mean fish, amphibians, reptiles, birds, andmammals, such as mice, rats, rabbits, goats, cats, dogs, cows, apes andhumans.

b. Attached

“Attached” or “immobilized” as used herein to refer to a probe and asolid support may mean that the binding between the probe and the solidsupport is sufficient to be stable under conditions of binding, washing,analysis, and removal. The binding may be covalent or non-covalent.Covalent bonds may be formed directly between the probe and the solidsupport or may be formed by a cross linker or by inclusion of a specificreactive group on either the solid support or the probe or bothmolecules. Non-covalent binding may be one or more of electrostatic,hydrophilic, and hydrophobic interactions. Included in non-covalentbinding is the covalent attachment of a molecule, such as streptavidin,to the support and the non-covalent binding of a biotinylated probe tothe streptavidin. Immobilization may also involve a combination ofcovalent and non-covalent interactions.

c. Biological Sample

“Biological sample” as used herein may mean a sample of biologicaltissue or fluid that comprises nucleic acids. Such samples include, butare not limited to, tissue isolated from animals. Biological samples mayalso include sections of tissues such as biopsy and autopsy samples,frozen sections taken for histologic purposes, blood, plasma, serum,sputum, stool, tears, mucus, hair, and skin. Biological samples alsoinclude explants and primary and/or transformed cell cultures derivedfrom patient tissues. A biological sample may be provided by removing asample of cells from an animal, but can also be accomplished by usingpreviously isolated cells (e.g., isolated by another person, at anothertime, and/or for another purpose), or by performing the methods of theinvention in vivo. Archival tissues, such as those having treatment oroutcome history, may also be used.

d. Complement

“Complement” or “complementary” as used herein may mean Watson-Crick orHoogsteen base pairing between nucleotides or nucleotide analogs ofnucleic acid molecules.

e. Differential Expression

“Differential expression” may mean qualitative or quantitativedifferences in the temporal and/or cellular gene expression patternswithin and among cells and tissue. Thus, a differentially expressed genecan qualitatively have its expression altered, including an activationor inactivation, in, e.g., normal versus disease tissue. Genes may beturned on or turned off in a particular state, relative to another statethus permitting comparison of two or more states. A qualitativelyregulated gene will exhibit an expression pattern within a state or celltype which may be detectable by standard techniques. Some genes will beexpressed in one state or cell type, but not in both. Alternatively, thedifference in expression may be quantitative, e.g., in that expressionis modulated, either up-regulated, resulting in an increased amount oftranscript, or down-regulated, resulting in a decreased amount oftranscript. The degree to which expression differs need only be largeenough to quantify via standard characterization techniques such asexpression arrays, quantitative reverse transcriptase PCR, northernanalysis, and RNase protection.

f. Gene

“Gene” used herein may be a genomic gene comprising transcriptionaland/or translational regulatory sequences and/or a coding region and/ornon-translated sequences (e.g., introns, 5′- and 3′-untranslatedsequences). The coding region of a gene may be a nucleotide sequencecoding for an amino acid sequence or a functional RNA, such as tRNA,rRNA, catalytic RNA, siRNA, miRNA and antisense RNA. A gene may also bean mRNA or cDNA corresponding to the coding regions (e.g., exons andmiRNA) optionally comprising 5′- or 3′-untranslated sequences linkedthereto. A gene may also be an amplified nucleic acid molecule producedin vitro comprising all or a part of the coding region and/or 5′- or3′-untranslated sequences linked thereto.

g. Host Cell

“Host cell” used herein may be a naturally occurring cell or atransformed cell that contains a vector and supports the replication ofthe vector. Host cells may be cultured cells, explants, cells in vivo,and the like. Host cells may be prokaryotic cells such as E. coli, oreukaryotic cells such as yeast, insect, amphibian, or mammalian cells,such as CHO, HeLa.

h. Identity

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences, may mean that the sequences havea specified percentage of nucleotides or amino acids that are the sameover a specified region. The percentage may be calculated by comparingoptimally aligning the two sequences, comparing the two sequences overthe specified region, determining the number of positions at which theidentical residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the specified region, and multiplying the resultby 100 to yield the percentage of sequence identity. In cases where thetwo sequences are of different lengths or the alignment producesstaggered end and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) are considered equivalent. Identitymay be performed manually or by using computer sequence algorithm suchas BLAST or BLAST 2.0.

i. Label

“Label” as used herein may mean a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and other entitieswhich can be made detectable. A label may be incorporated into nucleicacids and proteins at any position.

j. Nucleic Acid

“Nucleic acid” or “oligonucleotide” or “polynucleotide” used herein maymean at least two nucleotides covalently linked together. As will beappreciated by those in the art, the depiction of a single strand alsodefines the sequence of the complementary strand. Thus, a nucleic acidalso encompasses the complementary strand of a depicted single strand.As will also be appreciated by those in the art, many variants of anucleic acid may be used for the same purpose as a given nucleic acid.Thus, a nucleic acid also encompasses substantially identical nucleicacids and complements thereof. As will also be appreciated by those inthe art, a single strand provides a probe for a probe that may hybridizeto the target sequence under stringent hybridization conditions. Thus, anucleic acid also encompasses a probe that hybridizes under stringenthybridization conditions.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequence. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids may be obtained by chemical synthesismethods or by recombinant methods.

A nucleic acid will generally contain phosphodiester bonds, althoughnucleic acid analogs may be included that may have at least onedifferent linkage, e.g., phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphosphoroamidite linkages and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, which are incorporated by reference. Nucleic acids containingone or more non-naturally occurring or modified nucleotides are alsoincluded within one definition of nucleic acids. The modified nucleotideanalog may be located for example at the 5′-end and/or the 3′-end of thenucleic acid molecule. Representative examples of nucleotide analogs maybe selected from sugar- or backbone-modified ribonucleotides. It shouldbe noted, however, that also nucleobase-modified ribonucleotides, i.e.ribonucleotides, containing a non-naturally occurring nucleobase insteadof a naturally occurring nucleobase such as uridines or cytidinesmodified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromouridine; adenosines and guanosines modified at the 8-position, e.g.8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- andN-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The2′-OH-group may be replaced by a group selected from H, OR, R, halo, SH,SR, NH₂, NHR, NR₂ or CN, wherein R is C₁-C₆ alkyl, alkenyl or alkynyland halo is F, Cl, Br or I. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g., to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs may be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made.

k. Operably Linked

“Operably linked” used herein may mean that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter may be positioned 5′ (upstream) or 3′ (downstream) of the geneunder its control. The distance between the promoter and the gene may beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance can be accommodated withoutloss of promoter function.

l. Probe

“Probe” as used herein may mean an oligonucleotide capable of binding toa target nucleic acid of complementary sequence through one or moretypes of chemical bonds, usually through complementary base pairing,usually through hydrogen bond formation. Probes may bind targetsequences lacking complete complementarity with the probe sequencedepending upon the stringency of the hybridization conditions. There maybe any number of base pair mismatches which will interfere withhybridization between the target sequence and the single strandednucleic acids of the present invention. However, if the number ofmutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. A probe may be single stranded orpartially single and partially double stranded. The strandedness of theprobe is dictated by the structure, composition, and properties of thetarget sequence. Probes may be directly labeled or indirectly labeledsuch as with biotin to which a streptavidin complex may later bind.

m. Promoter

“Promoter” as used herein may mean a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter may comprise one ormore specific regulatory elements to further enhance expression and/orto alter the spatial expression and/or temporal expression of same. Apromoter may also comprise distal enhancer or repressor elements, whichcan be located as much as several thousand base pairs from the startsite of transcription. A promoter may be derived from sources includingviral, bacterial, fungal, plants, insects, and animals. A promoter mayregulate the expression of a gene component constitutively, ordifferentially with respect to cell, the tissue or organ in whichexpression occurs or, with respect to the developmental stage at whichexpression occurs, or in response to external stimuli such asphysiological stresses, pathogens, metal ions, or inducing agents.Representative examples of promoters include the bacteriophage T7promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

n. Selectable Marker

“Selectable marker” used herein may mean any gene which confers aphenotype on a cell in which it is expressed to facilitate theidentification and/or selection of cells which are transfected ortransformed with a genetic construct. Representative examples ofselectable markers include the ampicillin-resistance gene (Amp^(r)),tetracycline-resistance gene (Tc^(r)), bacterial kanamycin-resistancegene (Kan^(r)), zeocin resistance gene, the AURI-C gene which confersresistance to the antibiotic aureobasidin A, phosphinothricin-resistancegene, neomycin phosphotransferase gene (nptII), hygromycin-resistancegene, beta-glucuronidase (GUS) gene, chloramphenicol acetyltransferase(CAT) gene, green fluorescent protein-encoding gene and luciferase gene.

o. Stringent Hybridization Conditions

“Stringent hybridization conditions” used herein may mean conditionsunder which a first nucleic acid sequence (e.g., probe) will hybridizeto a second nucleic acid sequence (e.g., target), such as in a complexmixture of nucleic acids, but to no other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Generally, stringent conditions are selected to be about5-10° C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength pH. The T_(m) may be thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal may be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

p. Substantially Complementary

“Substantially complementary” used herein may mean that a first sequenceis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to the complement of a second sequence over a region of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50 or more nucleotides, or that the two sequences hybridizeunder stringent hybridization conditions.

q. Substantially Identical

“Substantially identical” used herein may mean that a first and secondsequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotidesor amino acids, or with respect to nucleic acids, if the first sequenceis substantially complementary to the complement of the second sequence.

r. Target

“Target” as used herein may mean a polynucleotide that may be bound byone or more probes under stringent hybridization conditions.

s. Terminator

“Terminator” used herein may mean a sequence at the end of atranscriptional unit which signals termination of transcription. Aterminator may be a 3′-non-translated DNA sequence containing apolyadenylation signal, which may facilitate the addition ofpolyadenylate sequences to the 3′-end of a primary transcript. Aterminator may be derived from sources including viral, bacterial,fungal, plants, insects, and animals. Representative examples ofterminators include the SV40 polyadenylation signal, HSV TKpolyadenylation signal, CYC1 terminator, ADH terminator, SPA terminator,nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens,the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zeingene terminator from Zea mays, the Rubisco small subunit gene (SSU) geneterminator sequences, subclover stunt virus (SCSV) gene sequenceterminators, rho-independent E. coli terminators, and the lacZ alphaterminator.

t. Vector

“Vector” used herein may mean a nucleic acid sequence containing anorigin of replication. A vector may be a plasmid, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectormay be a DNA or RNA vector. A vector may be either a self-replicatingextrachromosomal vector or a vector which integrate into a host genome.

2. MicroRNA

While not being bound by theory, the current model for the maturation ofmammalian miRNAs is shown in FIG. 1. A gene coding for a miRNA may betranscribed leading to production of an miRNA precursor known as thepri-miRNA. The pri-miRNA may be part of a polycistronic RNA comprisingmultiple pri-miRNAs. The pri-miRNA may form a hairpin with a stem andloop. As indicated on FIG. 1, the stem may comprise mismatched bases.

The hairpin structure of the pri-miRNA may be recognized by Drosha,which is an RNase III endonuclease. Drosha may recognize terminal loopsin the pri-miRNA and cleave approximately two helical turns into thestem to produce a 60-70 nt precursor known as the pre-miRNA. Drosha maycleave the pri-miRNA with a staggered cut typical of RNase IIIendonucleases yielding a pre-miRNA stem loop with a 5′ phosphate and ˜2nucleotide 3′ overhang. Approximately one helical turn of stem (˜10nucleotides) extending beyond the Drosha cleavage site may be essentialfor efficient processing. The pre-miRNA may then be actively transportedfrom the nucleus to the cytoplasm by Ran-GTP and the export receptorEx-portin-5.

The pre-miRNA may be recognized by Dicer, which is also an RNase IIIendonuclease. Dicer may recognize the double-stranded stem of thepre-miRNA. Dicer may also recognize the 5′ phosphate and 3′ overhang atthe base of the stem loop. Dicer may cleave off the terminal loop twohelical turns away from the base of the stem loop leaving an additional5′ phosphate and ˜2 nucleotide 3′ overhang. The resulting siRNA-likeduplex, which may comprise mismatches, comprises the mature miRNA and asimilar-sized fragment known as the miRNA*. The miRNA and miRNA* may bederived from opposing arms of the pri-miRNA and pre-miRNA. MiRNA*sequences may be found in libraries of cloned miRNAs but typically atlower frequency than the miRNAs.

Although initially present as a double-stranded species with miRNA*, themiRNA may eventually become incorporated as single-stranded RNAs into aribonucleoprotein complex known as the RNA-induced silencing complex(RISC). Various proteins can form the RISC, which can lead tovariability in specificity for miRNA/miRNA* duplexes, binding site ofthe target gene, activity of miRNA (repress or activate), which strandof the miRNA/miRNA* duplex is loaded in to the RISC.

When the miRNA strand of the miRNA:miRNA* duplex is loaded into theRISC, the miRNA* may be removed and degraded. The strand of themiRNA:miRNA* duplex that is loaded into the RISC may be the strand whose5′ end is less tightly paired. In cases where both ends of themiRNA:miRNA* have roughly equivalent 5′ pairing, both miRNA and miRNA*may have gene silencing activity.

The RISC may identify target nucleic acids based on high levels ofcomplementarity between the miRNA and the mRNA, especially bynucleotides 2-8 of the miRNA. Only one case has been reported in animalswhere the interaction between the miRNA and its target was along theentire length of the miRNA. This was shown for mir-196 and Hox B8 and itwas further shown that mir-196 mediates the cleavage of the Hox B8 mRNA(Yekta et al 2004, Science 304-594). Otherwise, such interactions areknown only in plants (Bartel & Bartel 2003, Plant Physiol 132-709).

A number of studies have looked at the base-pairing requirement betweenmiRNA and its mRNA target for achieving efficient inhibition oftranslation (reviewed by Bartel 2004, Cell 116-281). In mammalian cells,the first 8 nucleotides of the miRNA may be important (Doench & Sharp2004 Genes Dev 2004-504). However, other parts of the microRNA may alsoparticipate in mRNA binding. Moreover, sufficient base pairing at the 3′can compensate for insufficient pairing at the 5′ (Brennecke at al, 2005PLoS 3-e85). Computation studies, analyzing miRNA binding on wholegenomes have suggested a specific role for bases 2-7 at the 5′ of themiRNA in target binding but the role of the first nucleotide, foundusually to be “A” was also recognized (Lewis et at 2005 Cell 120-15).Similarly, nucleotides 1-7 or 2-8 were used to identify and validatetargets by Krek et al (2005, Nat Genet 37-495).

The target sites in the mRNA may be in the 5′ UTR, the 3′ UTR or in thecoding region. Interestingly, multiple miRNAs may regulate the same mRNAtarget by recognizing the same or multiple sites. The presence ofmultiple miRNA complementarity sites in most genetically identifiedtargets may indicate that the cooperative action of multiple RISCsprovides the most efficient translational inhibition.

MiRNAs may direct the RISC to downregulate gene expression by either oftwo mechanisms: mRNA cleavage or translational repression. The miRNA mayspecify cleavage of the mRNA if the mRNA has a certain degree ofcomplementarity to the miRNA. When a miRNA guides cleavage, the cut maybe between the nucleotides pairing to residues 10 and 11 of the miRNA.Alternatively, the miRNA may repress translation if the miRNA does nothave the requisite degree of complementarity to the miRNA. Translationalrepression may be more prevalent in animals since animals may have alower degree of complementarity.

It should be notes that there may be variability in the 5′ and 3′ endsof any pair of miRNA and miRNA*. This variability may be due tovariability in the enzymatic processing of Drosha and Dicer with respectto the site of cleavage. Variability at the 5′ and 3′ ends of miRNA andmiRNA* may also be due to mismatches in the stem structures of thepri-miRNA and pre-miRNA. The mismatches of the stem strands may lead toa population of different hairpin structures. Variability in the stemstructures may also lead to variability in the products of cleavage byDrosha and Dicer.

3. Nucleic Acid

The present invention relates to an isolated nucleic acid comprising anucleotide sequence referred to in SEQ ID NOS: 1-760616, the sequencesset forth on the Sequence Listing of U.S. patent application Ser. No.10/709,572, the contents of which are incorporated herein by reference,and the sequences set forth on the Sequence Listing of U.S. ProvisionalPatent Application No. 60/655,094, the contents of which areincorporated herein by reference, or variants thereof. The variant maybe a complement of the referenced nucleotide sequence. The variant mayalso be a nucleotide sequence that is substantially identical to thereferenced nucleotide sequence or the complement thereof. The variantmay also be a nucleotide sequence which hybridizes under stringentconditions to the referenced nucleotide sequence, complements thereof,or nucleotide sequences substantially identical thereto.

The nucleic acid may have a length of from 10 to 100 nucleotides. Thenucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,60, 70, 80 or 90 nucleotides. The nucleic acid may be synthesized orexpressed in a cell (in vitro or in vivo) using a synthetic genedescribed below. The nucleic acid may be synthesized as a single strandmolecule and hybridized to a substantially complementary nucleic acid toform a duplex, which is considered a nucleic acid of the invention. Thenucleic acid may be introduced to a cell, tissue or organ in a single-or double-stranded form or capable of being expressed by a syntheticgene using methods well known to those skilled in the art, including asdescribed in U.S. Pat. No. 6,506,559 which is incorporated by reference.

a. Pri-miRNA

The nucleic acid of the invention may comprise a sequence of a pri-miRNAor a variant thereof. The pri-miRNA sequence may comprise from 45-250,55-200, 70-150 or 80-100 nucleotides. The sequence of the pri-miRNA maycomprise a pre-miRNA, miRNA and miRNA* as set forth below. The pri-miRNAmay also comprise a miRNA or miRNA* and the complement thereof, andvariants thereof. The pri-miRNA may comprise at least 19% adenosinenucleotides, at least 16% cytosine nucleotides, at least 23% thyminenucleotides and at least 19% guanine nucleotides.

The pri-miRNA may form a hairpin structure. The hairpin may comprise afirst and second nucleic acid sequence that are substantiallycomplimentary. The first and second nucleic acid sequence may be from37-50 nucleotides. The first and second nucleic acid sequence may beseparated by a third sequence of from 8-12 nucleotides. The hairpinstructure may have a free energy less than −25 Kcal/mole as calculatedby the Vienna algorithm with default parameters, as described inHofacker et al., Monatshefte f. Chemie 125: 167-188 (1994), the contentsof which are incorporated herein. The hairpin may comprise a terminalloop of 4-20, 8-12 or 10 nucleotides.

The sequence of the pri-miRNA may comprise the sequence of a hairpinreferred to in Table 1, the sequence of sequence identifiers6757248-6894882 of the Sequence Listing of U.S. patent application Ser.No. 10/709,572, the contents of which are incorporated herein byreference, the sequence of sequence identifiers 1-6318 or 18728-18960 ofthe Sequence Listing of U.S. Provisional Patent Application No.60/655,094, the contents of which are incorporated herein by reference,or variants thereof.

b. Pre-miRNA

The nucleic acid of the invention may also comprise a sequence of apre-miRNA or a variant thereof. The pre-miRNA sequence may comprise from45-90, 60-80 or 60-70 nucleotides. The sequence of the pre-miRNA maycomprise a miRNA and a miRNA* as set forth below. The pre-miRNA may alsocomprise a miRNA or miRNA* and the complement thereof, and variantsthereof. The sequence of the pre-miRNA may also be that of a pri-miRNAexcluding from 0-160 nucleotides from the 5′ and 3′ ends of thepri-miRNA.

The sequence of the pre-miRNA may comprise the sequence of a hairpinreferred to in Table 1, the sequence of sequence identifiers6757248-6894882 of the Sequence Listing of U.S. patent application Ser.No. 10/709,572, the contents of which are incorporated herein byreference, the sequence of sequence identifiers 1-6318 or 18728-18960 ofthe Sequence Listing of U.S. Provisional Patent Application No.60/655,094, the contents of which are incorporated herein by reference,or variants thereof.

c. MiRNA

The nucleic acid of the invention may also comprise a sequence of amiRNA, miRNA* or a variant thereof. The miRNA sequence may comprise from13-33, 18-24 or 21-23 nucleotides. The sequence of the miRNA may be thefirst 13-33 nucleotides of the pre-miRNA. The sequence of the miRNA maybe the last 13-33 nucleotides of the pre-miRNA.

The sequence of the miRNA may comprise the sequence of a miRNA referredto in Table 1, the sequence of sequence identifiers 1-117750 or6894883-10068177 of the Sequence Listing of U.S. patent application Ser.No. 10/709,572, the contents of which are incorporated herein byreference, the sequence of sequence identifiers 6319-18727 or18961-19401 of the Sequence Listing of U.S. Provisional PatentApplication No. 60/655,094, the contents of which are incorporatedherein by reference, or variants thereof.

d. Anti-miRNA

The nucleic acid of the invention may also comprise a sequence of ananti-miRNA that is capable of blocking the activity of a miRNA ormiRNA*. The anti-miRNA may comprise a total of 5-100 or 10-60nucleotides. The anti-miRNA may also comprise a total of at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25or 26 nucleotides. The sequence of the anti-miRNA may comprise (a) atleast 5 nucleotides that are substantially identical to the 5′ of amiRNA and at least 5-12 nucleotide that are substantially complimentaryto the flanking regions of the target site from the 5′ end of saidmiRNA, or (b) at least 5-12 nucleotides that are substantially identicalto the 3′ of a miRNA and at least 5 nucleotide that are substantiallycomplimentary to the flanking region of the target site from the 3′ endof said miRNA.

The sequence of the anti-miRNA may comprise the compliment of a sequenceof a miRNA referred to in Table 1, the sequence of sequence identifiers1-117750 or 6894883-10068177 of the Sequence Listing of U.S. patentapplication Ser. No. 10/709,572, the contents of which are incorporatedherein by reference, the sequence of sequence identifiers 6319-18727 or18961-19401 of the Sequence Listing of U.S. Provisional PatentApplication No. 60/655,094, the contents of which are incorporatedherein by reference, or variants thereof.

e. Binding Site of Target

The nucleic acid of the invention may also comprise a sequence of atarget miRNA binding site, or a variant thereof. The target sitesequence may comprise a total of 5-100 or 10-60 nucleotides. The targetsite sequence may comprise at least 5 nucleotides of the sequence of atarget gene binding site referred to in Table 4, the sequence ofsequence identifiers 117751-6757247 of the Sequence Listing of U.S.patent application Ser. No. 10/709,572, the contents of which areincorporated herein by reference, or variants thereof.

4. Synthetic Gene

The present invention also relates to a synthetic gene comprising anucleic acid of the invention operably linked to a transcriptionaland/or translational regulatory sequences. The synthetic gene may becapable of modifying the expression of a target gene with a binding sitefor the nucleic acid of the invention. Expression of the target gene maybe modified in a cell, tissue or organ. The synthetic gene may besynthesized or derived from naturally-occurring genes by standardrecombinant techniques. The synthetic gene may also comprise terminatorsat the 3′-end of the transcriptional unit of the synthetic genesequence. The synthetic gene may also comprise a selectable marker.

5. Vector

The present invention also relates to a vector comprising a syntheticgene of the invention. The vector may be an expression vector. Anexpression vector may comprise additional elements. For example, theexpression vector may have two replication systems allowing it to bemaintained in two organisms, e.g., in mammalian or insect cells forexpression and in a prokaryotic host for cloning and amplification. Forintegrating expression vectors, the expression vector may contain atleast one sequence homologous to the host cell genome, and preferablytwo homologous sequences which flank the expression construct. Theintegrating vector may be directed to a specific locus in the host cellby selecting the appropriate homologous sequence for inclusion in thevector. The vector may also comprise a selectable marker gene to allowthe selection of transformed host cells.

6. Host Cell

The present invention also relates to a host cell comprising a vector ofthe invention. The cell may be a bacterial, fungal, plant, insect oranimal cell.

7. Probes

The present invention also relates to a probe comprising a nucleic acidof the invention. Probes may be used for screening and diagnosticmethods, as outlined below. The probe may be attached or immobilized toa solid substrate, such as a biochip.

The probe may have a length of from 8 to 500, 10 to 100 or 20 to 60nucleotides. The probe may also have a length of at least 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220,240, 260, 280 or 300 nucleotides. The probe may further comprise alinker sequence of from 10-60 nucleotides.

8. Biochip

The present invention also relates to a biochip. The biochip maycomprise a solid substrate comprising an attached probe or plurality ofprobes of the invention. The probes may be capable of hybridizing to atarget sequence under stringent hybridization conditions. The probes maybe attached at spatially defined address on the substrate. More than oneprobe per target sequence may be used, with either overlapping probes orprobes to different sections of a particular target sequence. The probesmay be capable of hybridizing to target sequences associated with asingle disorder.

The probes may be attached to the biochip in a wide variety of ways, aswill be appreciated by those in the art. The probes may either besynthesized first, with subsequent attachment to the biochip, or may bedirectly synthesized on the biochip.

The solid substrate may be a material that may be modified to containdiscrete individual sites appropriate for the attachment or associationof the probes and is amenable to at least one detection method.Representative examples of substrates include glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses andplastics. The substrates may allow optical detection without appreciablyfluorescing.

The substrate may be planar, although other configurations of substratesmay be used as well. For example, probes may be placed on the insidesurface of a tube, for flow-through sample analysis to minimize samplevolume. Similarly, the substrate may be flexible, such as a flexiblefoam, including closed cell foams made of particular plastics.

The biochip and the probe may be derivatized with chemical functionalgroups for subsequent attachment of the two. For example, the biochipmay be derivatized with a chemical functional group including, but notlimited to, amino groups, carboxyl groups, oxo groups or thiol groups.Using these functional groups, the probes may be attached usingfunctional groups on the probes either directly or indirectly using alinkers. The probes may be attached to the solid support by either the5′ terminus, 3′ terminus, or via an internal nucleotide.

The probe may also be attached to the solid support non-covalently. Forexample, biotinylated oligonucleotides can be made, which may bind tosurfaces covalently coated with streptavidin, resulting in attachment.Alternatively, probes may be synthesized on the surface using techniquessuch as photopolymerization and photolithography.

9. miRNA Expression Analysis

The present invention also relates to a method of identifying miRNAsthat are associated with disease or a pathological condition comprisingcontacting a biological sample with a probe or biochip of the inventionand detecting the amount of hybridization. PCR may be used to amplifynucleic acids in the sample, which may provide higher sensitivity.

The ability to identify miRNAs that are overexpressed or underexpressedin pathological cells compared to a control can provide high-resolution,high-sensitivity datasets which may be used in the areas of diagnostics,therapeutics, drug development, pharmacogenetics, biosensor development,and other related areas. An expression profile generated by the currentmethods may be a “fingerprint” of the state of the sample with respectto a number of miRNAs. While two states may have any particular miRNAsimilarly expressed, the evaluation of a number of miRNAs simultaneouslyallows the generation of a gene expression profile that ischaracteristic of the state of the cell. That is, normal tissue may bedistinguished from diseased tissue. By comparing expression profiles oftissue in known different disease states, information regarding whichmiRNAs are associated in each of these states may be obtained. Then,diagnosis may be performed or confirmed to determine whether a tissuesample has the expression profile of normal or disease tissue. This mayprovide for molecular diagnosis of related conditions.

10. Determining Expression Levels

The present invention also relates to a method of determining theexpression level of a disease-associated miRNA comprising contacting abiological sample with a probe or biochip of the invention and measuringthe amount of hybridization. The expression level of adisease-associated miRNA is information in a number of ways. Forexample, a differential expression of a disease-associated miRNAcompared to a control may be used as a diagnostic that a patient suffersfrom the disease. Expression levels of a disease-associated miRNA mayalso be used to monitor the treatment and disease state of a patient.Furthermore, expression levels of e disease-associated miRNA may allowthe screening of drug candidates for altering a particular expressionprofile or suppressing an expression profile associated with disease.

A target nucleic acid may be detected by contacting a sample comprisingthe target nucleic acid with a biochip comprising an attached probesufficiently complementary to the target nucleic acid and detectinghybridization to the probe above control levels.

The target nucleic acid may also be detected by immobilizing the nucleicacid to be examined on a solid support such as nylon membranes andhybridizing a labelled probe with the sample. Similarly, the targetnucleic may also be detected by immobilizing the labeled probe to thesolid support and hybridizing a sample comprising a labeled targetnucleic acid. Following washing to remove the non-specifichybridization, the label may be detected.

The target nucleic acid may also be detected in situ by contactingpermeabilized cells or tissue samples with a labeled probe to allowhybridization with the target nucleic acid. Following washing to removethe non-specifically bound probe, the label may be detected.

These assays can be direct hybridization assays or can comprise sandwichassays, which include the use of multiple probes, as is generallyoutlined in U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117;5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802;5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681,697, each of whichis hereby incorporated by reference.

A variety of hybridization conditions may be used, including high,moderate and low stringency conditions as outlined above. The assays maybe performed under stringency conditions which allow hybridization ofthe probe only to the target. Stringency can be controlled by altering astep parameter that is a thermodynamic variable, including, but notlimited to, temperature, formamide concentration, salt concentration,chaotropic salt concentration pH, or organic solvent concentration.

Hybridization reactions may be accomplished in a variety of ways.Components of the reaction may be added simultaneously, or sequentially,in different orders. In addition, the reaction may include a variety ofother reagents. These include salts, buffers, neutral proteins, e.g.,albumin, detergents, etc. which may be used to facilitate optimalhybridization and detection, and/or reduce non-specific or backgroundinteractions. Reagents that otherwise improve the efficiency of theassay, such as protease inhibitors, nuclease inhibitors andanti-microbial agents may also be used as appropriate, depending on thesample preparation methods and purity of the target.

a. Diagnostic

The present invention also relates to a method of diagnosis comprisingdetecting a differential expression level of a disease-associated miRNAin a biological sample. The sample may be derived from a patient.Diagnosis of a disease state in a patient allows for prognosis andselection of therapeutic strategy. Further, the developmental stage ofcells may be classified by determining temporarily expressedmiRNA-molecules.

In situ hybridization of labeled probes to tissue arrays may beperformed. When comparing the fingerprints between an individual and astandard, the skilled artisan can make a diagnosis, a prognosis, or aprediction based on the findings. It is further understood that thegenes which indicate the diagnosis may differ from those which indicatethe prognosis and molecular profiling of the condition of the cells maylead to distinctions between responsive or refractory conditions or maybe predictive of outcomes.

b. Drug Screening

The present invention also relates to a method of screening therapeuticscomprising contacting a pathological cell capable of expressing adisease related miRNA with a candidate therapeutic and evaluating theeffect of a drug candidate on the expression profile of the diseaseassociated miRNA. Having identified the differentially expressed miRNAs,a variety of assays may be executed. Test compounds may be screened forthe ability to modulate gene expression of the disease associated miRNA.Modulation includes both an increase and a decrease in gene expression.

The test compound or drug candidate may be any molecule, e.g., protein,oligopeptide, small organic molecule, polysaccharide, polynucleotide,etc., to be tested for the capacity to directly or indirectly alter thedisease phenotype or the expression of the disease associated miRNA.Drug candidates encompass numerous chemical classes, such as smallorganic molecules having a molecular weight of more than 100 and lessthan about 500, 1,000, 1,500, 2,000 or 2,500 daltons. Candidatecompounds may comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Combinatorial libraries of potential modulators may be screened for theability to bind to the disease associated miRNA or to modulate theactivity thereof. The combinatorial library may be a collection ofdiverse chemical compounds generated by either chemical synthesis orbiological synthesis by combining a number of chemical building blockssuch as reagents. Preparation and screening of combinatorial chemicallibraries is well known to those of skill in the art. Such combinatorialchemical libraries include, but are not limited to, peptide librariesencoded peptides, benzodiazepines, diversomers such as hydantoins,benzodiazepines and dipeptide, vinylogous polypeptides, analogousorganic syntheses of small compound libraries, oligocarbamates, and/orpeptidyl phosphonates, nucleic acid libraries, peptide nucleic acidlibraries, antibody libraries, carbohydrate libraries, and small organicmolecule libraries.

11. Gene Silencing

The present invention also relates to a method of using the nucleicacids of the invention to reduce expression of a target gene in a cell,tissue or organ. Expression of the target gene may be reduced byexpressing a nucleic acid of the invention that comprises a sequencesubstantially complementary to one or more binding sites of the targetmRNA. The nucleic acid may be a miRNA or a variant thereof. The nucleicacid may also be pri-miRNA, pre-miRNA, or a variant thereof, which maybe processed to yield a miRNA. The expressed miRNA may hybridize to asubstantially complementary binding site on the target mRNA, which maylead to activation of RISC-mediated gene silencing. An example for astudy employing over-expression of miRNA is Yekta et al 2004, Science304-594, which is incorporated herein by reference. One of ordinaryskill in the art will recognize that the nucleic acids of the presentinvention may be used to inhibit expression of target genes usingantisense methods well known in the art, as well as RNAi methodsdescribed in U.S. Pat. Nos. 6,506,559 and 6,573,099, which areincorporated by reference.

The target of gene silencing may be a protein that causes the silencingof a second protein. By repressing expression of the target gene,expression of the second protein may be increased. Examples forefficient suppression of miRNA expression are the studies by Esau et al2004 JBC 275-52361; and Cheng et al 2005 Nucleic Acids Res. 33-1290,which is incorporated herein by reference.

12. Gene Enhancement

The present invention also relates to a method of using the nucleicacids of the invention to increase expression of a target gene in acell, tissue or organ. Expression of the target gene may be increased byexpressing a nucleic acid of the invention that comprises a sequencesubstantially complementary to a pri-miRNA, pre-miRNA, miRNA or avariant thereof. The nucleic acid may be an anti-miRNA. The anti-miRNAmay hybridize with a pri-miRNA, pre-miRNA or miRNA, thereby reducing itsgene repression activity. Expression of the target gene may also beincreased by expressing a nucleic acid of the invention that issubstantially complementary to a portion of the binding site in thetarget gene, such that binding of the nucleic acid to the binding sitemay prevent miRNA binding.

13. Therapeutic

The present invention also relates to a method of using the nucleicacids of the invention as modulators or targets of disease or disordersassociated with developmental dysfunctions, such as cancer. In general,the claimed nucleic acid molecules may be used as a modulator of theexpression of genes which are at least partially complementary to saidnucleic acid. Further, miRNA molecules may act as target for therapeuticscreening procedures, e.g. inhibition or activation of miRNA moleculesmight modulate a cellular differentiation process, e.g. apoptosis.

Furthermore, existing miRNA molecules may be used as starting materialsfor the manufacture of sequence-modified miRNA molecules, in order tomodify the target-specificity thereof, e.g. an oncogene, amultidrug-resistance gene or another therapeutic target gene. Further,miRNA molecules can be modified, in order that they are processed andthen generated as double-stranded siRNAs which are again directedagainst therapeutically relevant targets. Furthermore, miRNA moleculesmay be used for tissue reprogramming procedures, e.g. a differentiatedcell line might be transformed by expression of miRNA molecules into adifferent cell type or a stem cell.

14. Compositions

The present invention also relates to a pharmaceutical compositioncomprising the nucleic acids of the invention and optionally apharmaceutically acceptable carrier. The compositions may be used fordiagnostic or therapeutic applications. The administration of thepharmaceutical composition may be carried out by known methods, whereina nucleic acid is introduced into a desired target cell in vitro or invivo. Commonly used gene transfer techniques include calcium phosphate,DEAE-dextran, electroporation, microinjection, viral methods andcationic liposomes.

15. Kits

The present invention also relates to kits comprising a nucleic acid ofthe invention together with any or all of the following: assay reagents,buffers, probes and/or primers, and sterile saline or anotherpharmaceutically acceptable emulsion and suspension base. In addition,the kits may include instructional materials containing directions(e.g., protocols) for the practice of the methods of this invention.

Example 1 Prediction of miRNAs

We surveyed the entire human genome for potential miRNA coding genesusing two computational approaches similar to those described in U.S.Patent Application No. 60/522,459, Ser. Nos. 10/709,577 and 10/709,572,the contents of which are incorporated herein by reference, forpredicting miRNAs. Briefly, non-protein coding regions of the entirehuman genome were scanned for hairpin structures. The predicted hairpinsand potential miRNAs were scored by thermodynamic stability, as well asstructural and contextual features. The algorithm was calibrated byusing miRNAs in the Sanger Database which had been validated.

1. First Screen

Table 2 of U.S. patent application Ser. No. 10/709,572, the contents ofwhich are incorporated herein by reference, shows the sequence(“PRECURSOR SEQUENCE”), sequence identifier (“PRECUR SEQ-ID”) andorganism of origin (“GAM ORGANISM”) for each predicted hairpin from thefirst computational screen, together with the predicted miRNAs (“GAMNAME”). Table 1 of U.S. patent application Ser. No. 10/709,572, thecontents of which are incorporated herein by reference, shows thesequence (“GAM RNA SEQUENCE”) and sequence identifier (“GAM SEQ-ID”) foreach miRNA (“GAM NAME”), along with the organism of origin (“GAMORGANISM”) and Dicer cut location (“GAM POS”). The sequences of thepredicted hairpins and miRNA are also set forth on the Sequence Listingof U.S. patent application Ser. No. 10/709,572, the contents of whichare incorporated herein by reference.

2. Second Screen

Table 1 lists the SEQ ID NO for each predicted hairpin (“HID”) of thesecond computational screen. Table 1 also lists the genomic location foreach hairpin (“Hairpin Location”). The format for the genomic locationis a concatenation of <chr_id><strand><start position>. For example,19+135460000 refers chromosome 19, +strand, start position 135460000.Chromosomes 23-25 refer to chromosome X, chromosome Y and mitochondrialDNA. The chromosomal location is based on the hg17 assembly of the humangenome by UCSC, which is based on NCBI Build 35 version 1 and wasproduced by the International Human Genome Sequencing Consortium.

Table 1 also lists whether the hairpin is conserved in evolution (“C”).There is an option that there is a paper of the genome version. Thehairpins were identified as conserved (“Y”) or nonconserved (“N”) byusing phastCons data. The phastCons data is a measure of evolutionaryconservation for each nucleotide in the human genome against the genomesof chimp, mouse, rat, dog, chicken, frog, and zebrafish, based on aphylo-HMM using best-in-genome pair wise alignment for each speciesbased on BlastZ, followed by multiZ alignment of the 8 genomes (Siepelet al, J. Comput. Biol 11, 413-428, 2004 and Schwartz et al., GenomeRes. 13, 103-107, 2003). A hairpin is listed as conserved if the averagephastCons conservation score over the 7 species in any 15 nucleotidesequence within the hairpin stem is at least 0.9 (Berezikov, E. et al.Phylogenetic Shadowing and Computational Identification of HumanmicroRNA Genes. Cell 120, 21-24, 2005).

Table 1 also lists the genomic type for each hairpin (“T”) as eitherintergenic (“G”), intron (“I”) or exon (“E”). Table 1 also lists the SEQID NO (“MID”) for each predicted miRNA and miRNA*. Table 1 also liststhe prediction score grade for each hairpin (“P”) on a scale of 0-1 (1the hairpin is the most reliable), as described in Hofacker et al.,Monatshefte f. Chemie 125: 167-188, 1994. If the grade is zero or null,they are transformed to the lower value of PalGrade that its p-value is<0.05. Table 1 also lists the p-value (“Pval”) calculated out ofbackground hairpins for the values of each P scores. As shown in Table,there are few instances where the Pval is >0.05. In each of these cases,the hairpins are highly conserved or they have been validated (F=Y).

Table 1 also lists whether the miRNAs were validated by expressionanalysis (“E”) (Y=Yes, N=No), as detailed in Table 2. Table 1 also listswhether the miRNAs were validated by sequencing (“S”) (Y=Yes, N=No). Ifthere was a difference in sequences between the predicted and sequencedmiRNAs, the sequenced sequence is predicted. It should be noted thatfailure to sequence or detect expression of a miRNA does not necessarilymean that a miRNA does not exist. Such undetected miRNAs may beexpressed in tissues other than those tested. In addition, suchundetected miRNAs may be expressed in the test tissues, but at adifference stage or under different condition than those of theexperimental cells.

Table 1 also listed whether the miRNAs were shown to be differentiallyexpressed (“D”) (Y=Yes, N=No) in at least one disease, as detailed inTable 2). Table 1 also whether the miRNAs were present (“F”) (Y=Yes,N=No) in Sanger DB Release 6.0 (April 2005)(http://nar.oupjournals.org/) as being detected in humans or mice orpredicted in humans. As discussed above, the miRNAs listed in the Sangerdatabase are a component of the prediction algorithm and a control forthe output.

Table 1 also lists a genetic location cluster (“LC”) for those hairpinsthat are within 5,000 nucleotides of each other. Each miRNA that has thesame LC share the same genetic cluster. Table 1 also lists a seedcluster (“SC”) to group miRNAs by their seed of 2-7 by an exact match.Each miRNA that has the same SC have the same seed. For a discussion ofseed lengths of 5 nucleotides, see Lewis et al., Cell, 120; 15-20(2005).

The table below shows the information from Table 1 about the miRNAhaving SEQ ID NO: 8087 and hairpin having SEQ ID NO: 550.

HID Hairpin Loc C T MID P Pval E S D F LC SC 550 19+58982742 Y G 80870.57 0.0043 Y N N N 427 874

Example 2 Prediction of Target Genes

The predicted miRNAs from the two computational screens of Example 1were then used to predict target genes and their binding sites using twocomputational approaches similar to those described in U.S. PatentApplication No. 60/522,459, Ser. Nos. 10/709,577 and 10/709,572, thecontents of which are incorporated herein by reference, for predictingmiRNAs.

1. First Screen

Table 6 of U.S. patent application Ser. No. 10/709,572, the contents ofwhich are incorporated herein by reference, lists the predicted targetgenes (“TARGET”) and binding site sequence (“TARGET BINDING SITESEQUENCE”) and binding site sequence identifier (“TARGET BINDING SITESEQ-ID”) from the first computational screen, as well as the organism oforigin for the target (“TARGET ORGANISM”). Table 12 of U.S. patentapplication Ser. No. 10/709,572, the contents of which are incorporatedherein by reference, lists the diseases (“DISEASE NAME”) that areassociated with the target genes (“TARGET-GENES ASSOCIATED WITHDISEASE”). Table 14 of U.S. patent application Ser. No. 10/709,572, thecontents of which are incorporated herein by reference, lists thesequence identifiers for the miRNAs (“SEQ ID NOs OF GAMS ASSOCIATED WITHDISEASE”) and the diseases (“DISEASE NAME”) that are associated with themiRNA based on the target gene. The sequences of the binding sitesequences are also set forth on the Sequence Listing of U.S. patentapplication Ser. No. 10/709,572, the contents of which are incorporatedherein by reference.

2. Second Screen

Table 4 lists the predicted target gene for each miRNA (MID) and itshairpin (HID) from the second computational screen. The names of thetarget genes were taken from NCBI Reference Sequence release 9; Pruittet al., Nucleic Acids Res, 33(1):D501-D504, 2005; Pruitt et al., TrendsGenet., 16(1):44-47, 2000; and Tatusova et al., Bioinformatics,15(7-8):536-43, 1999). Target genes were identified by having a perfectcomplimentary match of a 7 nucleotide miRNA seed (positions 2-8) and anA on the UTR (total=8 nucleotides). For a discussion on identifyingtarget genes, see Lewis et al., Cell, 120: 15-20, (2005). For adiscussion of the seed being sufficient for binding of a miRNA to a UTR,see Lim Lau et al., (Nature 2005) and Brenneck et al, (PLoS Biol 2005).

Binding sites were then predicted using a filtered target genes datasetby including only those target genes that contained a UTR of a least 30nucleotides. The binding site screen only considered the first 4000nucleotides per UTR and considered the longest transcript when therewere several transcripts per gene. The filtering reduced the totalnumber of transcripts from 23626 to 14239. Table 4 lists the SEQ ID NOfor the predicted binding sites for each target gene. The sequence ofthe binding site includes the 20 nucleotides 5′ and 3′ of the bindingsite as they are located on the spliced mRNA. Except for those miRNAsthat have only a single predicted binding site or those miRNAs that werevalidated, the data in Table 4 has been filtered to only indicate thosetarget genes with at least 2 binding sites.

The table below shows the information from Table 4 about the miRNAhaving SEQ ID NO: 8087 and hairpin having SEQ ID NO: 550.

HID MID Target Genes and Binding Sites 550 8087 CARD8 (89113, 89114);CD28 (89099, 89100); CLEC5A (89105, 89106); DCBLD (89101, 89102); DPP10(89097, 89098); FEZ1 (89107, 89108); G3BP2 (89103 89104); KCTD12 (89109,89110); LRRC3 (89115, 89116); SEC14L1 (89111 89112); SEMA4F (89095,89096);

Table 5 shows the relationship between the miRNAs (“MID”)/hairpins(“HID”) and diseases by their target genes. The name of diseases aretaken from OMIM. For a discussion of the rational for connecting thehost gene the hairpin is located upon to disease, see Baskerville andBartel, RNA, 11: 241-247 (2005) and Rodriguez et al., Genome Res., 14:1902-1910 (2004). Table 5 shows the number of miRNA target genes (“N”)that are related to the disease. Table 5 also shows the total number ofgenes that are related to the disease (“T”), which is taken from thegenes that were predicted to have binding sites for miRNAs. Table 5 alsoshows the percentage of N out of T and the p-value of hypergeometricanalysis (“Pval”). Table 8 shows the disease codes for Tables 5 and 6.For a reference of hypergeometric analysis, see Schaum's Outline ofElements of Statistics II: Inferential Statistics.

Table 6 shows the relationship between the miRNAs (“MID”)/hairpins(“HID”) and diseases by their host genes. We defined hairpins genes onthe complementary strand of a host gene as located on the gene: Intron_cas Interon and Exon_c as Exon. We choose the complementary strands asthey can cause disease. For example, a mutation in the miRNA that islocated on the complementary strand. In those case that a miRNA in onboth strands, two statuses like when Intron and Exon_c Intron is the onechosen. The logic of choosing is Intron>Exon>Intron_c>Exon_c>Intergenic.Table 9 shows the relationship between the target sequences (“GeneName”) and disease (“Disease Code”).

Example 3 Validation of miRNAs 1. Expression Analysis—Set 1

To confirm the hairpins and miRNAs predicted in Example 1, we detectedexpression in various tissues using the high-throughput microarrayssimilar to those described in U.S. Patent Application No. 60/522,459,Ser. Nos. 10/709,577 and 10/709,572, the contents of which areincorporated herein by reference. For each predicted precursor miRNA,mature miRNAs derived from both stems of the hairpin were tested.

Table 2 shows the hairpins (“HID”) of the second prediction set thatwere validated by detecting expression of related miRNAs (“MID”), aswell as a code for the tissue (“Tissue”) that expression was detected.The tissue and diseases codes for Table 2 are listed in Table 7. Some ofthe tested tissues wee cell line. Lung carcinoma cell line (H1299)with/without P53: H1299 has a mutated P53. The cell line was transfectedwith a construct with P53 that is temperature sensitive (active at 32°C.). The experiment was conducted at 32° C.

Table 2 also shows the chip expression score grade (range of 500-65000).A threshold of 500 was used to eliminate non-significant signals and thescore was normalized by MirChip probe signals from differentexperiments. Variations in the intensities of fluorescence materialbetween experiments may be due to variability in RNA preparation orlabeling efficiency. We normalized based on the assumption that thetotal amount of miRNAs in each sample is relatively constant. First wesubtracted the background signal from the raw signal of each probe,where the background signal is defined as 400. Next, we divided eachmiRNA probe signal by the average signal of all miRNAs, multiplied theresult by 10000 and added back the background signal of 400. Thus, bydefinition, the sum of all miRNA probe signals in each experiment is10400.

Table 2 also shows a statistical analysis of the normalized signal(“Spval”) calculated on the normalized score. For each miRNA, we used arelevant control group out of the full predicted miRNA list. Each miRNAhas an internal control of probes with mismatches. The relevant controlgroup contained probes with similar C and G percentage (abs diff<5%) inorder to have similar Tm. The probe signal P value is the ratio over therelevant control group probes with the same or higher signals. Theresults are p-value ≦0.05 and score is above 500. In those cases thatthe SPVal is listed as 0.0, the value is less than 0.0001.

The table below shows the information from Table 2 about the miRNAhaving SEQ ID NO: 8087 and hairpin having SEQ ID NO: 550.

HID MID Tissue S SPval Disease R RPval 550 8087 8 862 0.0177 550 8087 1826689 0.0016

The following table shows the information from Table 7 about the miRNAhaving SEQ ID NO: 8087 and hairpin having SEQ ID NO: 550.

Tissue or Disease name ID Embryonic Stem cells 8 Embryonic Stemcarcinoma cells 18

2. Expression Analysis—Set 2

To further confirm the hairpins and miRNAs predicted in 0, we detectedexpression in additional tissues. Table 2 of U.S. Provisional PatentApplication No. 60/655,094, which is incorporated herein by reference,lists expression data of miRNAs by the following: HID: hairpin sequenceidentifier for sequence set forth in the Sequence Listing of U.S.Provisional Patent Application No. 60/655,094, which is incorporatedherein by reference; MID: miRNA sequence identifier for sequence setforth in the Sequence Listing of U.S. Provisional Patent Application No.60/655,094, which is incorporated herein by reference; Tissue: testedtissue; S: chip expression score grade (range=100-65000); Dis. Diff.Exp.: disease related differential expression and the tissue it wastested in; R: ratio of disease related expression (range=0.01-99.99);and abbreviations: Brain Mix A—a mixture of brain tissue that areaffected in Alzheimer; Brain Mix B—a mixture of all brain tissues; andBrain SN—Substantia Nigra.

3. Sequencing

To further validate the hairpins (“HID”) of the second prediction, anumber of miRNAs were validated by sequencing methods similar to thosedescribed in U.S. Patent Application No. 60/522,459, Ser. Nos.10/709,577 and 10/709,572, the contents of which are incorporated hereinby reference. Table 3 shows the hairpins (“HID”) that were validated bysequencing a miRNA (MID) in the indicated tissue (“Tissue”).

Example 4 MiRNAs of Chromosome 19

A group of the validated miRNAs from Example 3 were highly expressed inplacenta, have distinct sequence similarity, and are located in the samelocus on chromosome 19 (FIG. 2). These predicted miRNAs are spread alonga region of ˜100,000 nucleotides in the 19q13.42 locus. This genomicregion is devoid of protein-coding genes and seems to be intergenic.Further analysis of the genomic sequence, including a thoroughexamination of the output of our prediction algorithm, revealed manymore putative related miRNAs, and located mir-371, mir-372, and mir-373approximately 25,000 bp downstream to this region. Overall, 54 putativemiRNA precursors were identified in this region. The miRNA precursorscan be divided into four distinct types of related sequences (FIG. 2).About 75% of the miRNAs in the cluster are highly related and werelabeled as type A. Three other miRNA types, types B, C and D, arecomposed of 4, 2, and 2 precursors, respectively. An additional 3putative miRNA precursors (S1 to S3) have unrelated sequences.Interestingly, all miRNA precursors are in the same orientation as theneighboring mir-371, mir-372, and mir-373 miRNA precursors.

Further sequence analysis revealed that the majority of the A-typemiRNAs are embedded in a ˜600 bp region that is repeated 35 times in thecluster. The repeated sequence does not appear in other regions of thegenome and is conserved only in primates. The repeating unit is almostalways bounded by upstream and downstream Alu repeats. This is in sharpcontrast to the MC14-1 cluster which is extremely poor in Alu repeats.

FIG. 3-A shows a comparison of sequences of the 35 repeat unitscontaining the A-type miRNA precursors in human. The comparisonidentified two regions exhibiting the highest sequence similarity. Oneregion includes the A-type miRNA, located in the 3′ region of therepeat. The second region is located ˜100 nucleotides upstream to theA-type miRNA precursors. However, the second region does not show highsimilarity among the chimp repeat units while the region containing theA-type miRNA precursors does (FIG. 3-B).

Examination of the region containing the A-type repeats showed that the5′ region of the miRNAs encoded by the 5′ stem of the precursors (5pmiRNAs) seem to be more variable than other regions of the maturemiRNAs. This is matched by variability in the 3′ region of the maturemiRNAs derived from the 3′ stems (3p miRNAs). As expected, the loopregion is highly variable. The same phenomenon can also be observed inthe multiple sequence alignment of all 43 A-type miRNAs (FIG. 4).

The multiple sequence alignment presented in FIG. 4 revealed thefollowing findings with regards to the predicted mature miRNAs. The 5pmiRNAs can be divided into 3 blocks. Nucleotides 1 to 6 are C/T rich,relatively variable, and are marked in most miRNAs by a CTC motif innucleotides 3 to 5. Nucleotides 7 to 15 are A/G rich and apart fromnucleotides 7 and 8 are shared among most of the miRNAs. Nucleotides 16to 23 are C/T rich and are, again, conserved among the members. Thepredicted 3p miRNAs, in general, show a higher conservation among thefamily members. Most start with an AAA motif, but a few have a different5′ sequence that may be critical in their target recognition.Nucleotides 8 to 15 are C/T rich and show high conservation. The last 7nucleotides are somewhat less conserved but include a GAG motif innucleotides 17 to 19 that is common to most members.

Analysis of the 5′ region of the repeated units identified potentialhairpins. However, in most repeating units these hairpins were notpreserved and efforts to clone miRNAs from the highest scoring hairpinsfailed. There are 8 A-type precursors that are not found within a longrepeating unit. Sequences surrounding these precursors show nosimilarity to the A-type repeating units or to any other genomicsequence. For 5 of these A-type precursors there are Alu repeats locatedsignificantly closer downstream to the A-type sequence.

The other miRNA types in the cluster showed the followingcharacteristics. The four B group miRNAs are found in a repeated regionof ˜500 bp, one of which is located at the end of the cluster. The twoD-type miRNAs, which are ˜2000 nucleotides from each other, are locatedat the beginning of the cluster and are included in a duplicated regionof 1220 nucleotides. Interestingly, the two D-type precursors areidentical. Two of the three miRNAs of unrelated sequence, S1 and S2, arelocated just after the two D-type miRNAs, and the third is locatedbetween A34 and A35. In general, the entire ˜100,000 nucleotide regioncontaining the cluster is covered with repeating elements. This includesthe miRNA-containing repeating units that are specific to this regionand the genome wide repeat elements that are spread in the cluster inlarge numbers.

Example 5 Cloning of Predicted MiRNAs

To further validate the predicted miRNAs, a number of the miRNAsdescribed in Example 4 were cloned using methods similar to thosedescribed in U.S. Patent Application No. 60/522,459, Ser. Nos.10/709,577 and 10/709,572, the contents of which are incorporated hereinby reference. Briefly, a specific capture oligonucleotide was designedfor each of the predicted miRNAs. The oligonucleotide was used tocapture, clone, and sequence the specific miRNA from a placenta-derivedlibrary enriched for small RNAs.

We cloned 41 of the 43 A-type miRNAs, of which 13 miRNAs were notpresent on the original microarray but only computationally predicted,as well as the D-type miRNAs. For 11 of the predicted miRNA precursors,both 5p and 3p predicted mature miRNAs were present on the microarrayand in all cases both gave significant signals. Thus, we attempted toclone both 5′ and 3′ mature miRNAs in all cloning attempts. For 27 ofthe 43 cloned miRNA, we were able to clone miRNA derived from both 5′and 3′ stems. Since our cloning efforts were not exhaustive, it ispossible that more of the miRNA precursors encode both 5′ and 3′ maturemiRNAs.

Many of the cloned miRNAs have shown heterogeneity at the 3′ end asobserved in many miRNA cloning studies (Lagos-Quintana 2001, 2002, 2003)(Poy 2004). Interestingly, we also observed heterogeneity at the 5′ endfor a significant number of the cloned miRNAs. This heterogeneity seemedto be somewhat more prevalent in 5′-stem derived miRNAs (9) compared to3′-stem derived miRNAs (6). In comparison, heterogeneity at the 3′ endwas similar for both 3′ and 5′-stem derived miRNAs (19 and 13,respectively). The 5′ heterogeneity involved mainly addition of onenucleotide, mostly C or A, but in one case there was an addition of 3nucleotides. This phenomenon is not specific to the miRNAs in thechromosome 19 cluster. We have observed it for many additional clonedmiRNAs, including both known miRNAs as well as novel miRNAs from otherchromosomes (data not shown).

Example 6 Analysis of MiRNA Expression

To further examine the expression of the miRNAs of Example 4, we usedNorthern blot analysis to profile miRNA expression in several tissues.Northern blot analysis was performed using 40 μg of total RNA separatedon 13% denaturing polyacrylamide gels and using ³²P end labeledoligonucleotide probes. The oligonucleotide probe sequences were5′-ACTCTAAAGAGAAGCGCTTTGT-3′(SEQ ID NO:760617) (A19-3p, NCBI:HSA-MIR-RG-21) and 5′-ACCCACCAAAGAGAAGCACTTT-3′(SEQ ID NO:760618)(A24-3p, NCBI: HSA-MIR-RG-27). The miRNAs were expressed as ˜22nucleotide long RNA molecules with tissue specificity profile identicalto that observed in the microarray analysis (FIG. 5-A).

In order to determine how the MC19-1 cluster is transcribed. A survey ofthe ESTs in the region identified only one place that included ESTs withpoly-adenylation signal and poly-A tail. This region is located justdownstream to the A43 precursor. The only other region that had ESTswith poly-adenylation signal is located just after mir-373, suggestingthat mir-371,2,3 are on a separate transcript. We performed initialstudies focusing on the region around mir-A43 to ensure that the regionis indeed transcribed into poly-adenylated mRNA. RT-PCR experimentsusing primers covering a region of 3.5 kb resulted in obtaining theexpected fragment (FIG. 5-B). RT-PCR analysis was performed using 5 μgof placenta total RNA using oligo-dT as primer. The following primerswere used to amplify the transcripts: f1:5′-GTCCCTGTACTGGAACTTGAG-3′(SEQ ID NO:760619); f2:5′-GTGTCCCTGTACTGGAACGCA-3′(SEQ ID NO:760620); r1:5′-GCCTGGCCATGTCAGCTACG-3′(SEQ ID NO: 760621); r2:5′-TTGATGGGAGGCTAGTGTTTC-3′(SEQ ID NO: 760622); r3:5′-GACGTGGAGGCGTTCTTAGTC-3′(SEQ ID NO: 760623); and r4:5′-TGACAACCGTTGGGGATTAC-3′ (SEQ ID NO: 760624). The authenticity of thefragment was validated by sequencing. This region includes mir-A42 andmir-A43, which shows that both miRNAs are present on the same primarytranscript.

Further information on the transcription of the cluster came fromanalysis of the 77 ESTs located within it. We found that 42 of the ESTswere derived from placenta. As these ESTs are spread along the entirecluster, it suggested that the entire cluster is expressed in placenta.This observation is in-line with the expression profile observed in themicroarray analysis. Thus, all miRNAs in the cluster may beco-expressed, with the only exception being the D-type miRNAs which arethe only miRNAs to be expressed in HeLa cells. Interestingly, none ofthe 77 ESTs located in the region overlap the miRNA precursors in thecluster. This is in-line with the depletion of EST representation fromtranscripts processed by Drosha.

Examination of the microarray expression profile revealed that miRNAsD1/2, A12, A21, A22, and A34, have a somewhat different expressionprofile reflected as low to medium expression levels in several of theother tissues examined. This may be explained by alternative splicing ofthe transcript(s) encoding the miRNAs or by the presence of additionalpromoter(s) of different tissues specificity along the cluster.

Comparison of the expression of 3p and 5p mature miRNAs revealed thatboth are expressed for many miRNA precursors but in most cases atdifferent levels. For most pre-miRNAs the 3p miRNAs are expressed athigher levels then the 5p miRNAs. However, in 6 cases (mir-D1,2, mir-A1,mir-A8, mir-A12, mir-A17 and mir-A33) both 3p and 5p miRNAs wereexpressed at a similar level, and in one case (mir-A32) the 5p miRNA wasexpressed at higher levels then the 3p miRNA.

Example 7 Conservation

Comparison of the sequences from all four types of predicted miRNAs ofExample 4 to that of other species (chimp, macaque, dog, chicken, mouse,rat, drosophila, zebra-fish, fungi, c. elegans) revealed that all miRNAsin the cluster, and in fact the entire region, are not conserved beyondprimates. Interestingly, homologues of this region do not exist in anyother genomes examined, including mouse and rat. Thus, this is the firstmiRNA cluster that is specific to primates and not generally shared inmammals. Homology analysis between chimp and human show that all 35repeats carrying the A-type miRNAs are contiguous between the twospecies. Furthermore, the entire cluster seems to be identical betweenhuman and chimp. Thus, the multiple duplications leading to theemergence of the MC19-1 cluster must have occurred prior to the split ofchimp and human and remained stable during the evolution of eachspecies. It should be noted that human chromosome 19 is known to includemany tandemly clustered gene families and large segmental duplications(Grimwood et al, 2004). Thus, in this respect the MC19-1 cluster is anatural part of chromosome 19.

In comparison, the MC14-1 cluster is generally conserved in mouse andincludes only the A7 and A8 miRNAs within the cluster are not conservedbeyond primates (Seitz 2004). In contrast all miRNAs in the MC19-1cluster are unique to primates. A survey of all miRNAs found in Sangerrevealed that only three miRNA, mir-198, mir-373, and mir-422a, are notconserved in the mouse or rat genomes, however, they are conserved inthe dog genome and are thus not specific to primates. Interestingly,mir-371 and mir-372, which are clustered with mir-373, and are located25 kb downstream to the MC19-1 cluster, are homologous to some extent tothe A-type miRNAs (FIG. 4), but are conserved in rodents.

Comparison of the A-type miRNA sequences to the miRNAs in the Sangerdatabase revealed the greatest homology to the human mir-302 family(FIG. 4-C). This homology is higher than the homology observed withmir-371,2,3. The mir-302 family (mir-302a, b, c, and d) are found in atightly packed cluster of five miRNAs (including mir-367) covering 690nucleotides located in the antisense orientation in the first intronwithin the protein coding exons of the HDCMA18P gene (accessionNM_(—)016648). No additional homology, apart from the miRNA homology,exists between the mir-302 cluster and the MC19-1 cluster. The fact thatboth the mir-371,2,3 and mir-302a,b,c,d are specific to embryonic stemcells is noteworthy.

Example 8 Differential Expression of miRNAs

Using chip expression methods similar to those described in 0,microarray images were analyzed using Feature Extraction Software(Version 7.1.1, Agilent). Table 2 shows the ratio of disease relatedexpression (“R”) compared to normal tissues. Table 2 also shows thestatistical analysis of the normalized signal (“RPval”). The signal ofeach probe was set as its median intensity. Signal intensities rangefrom background level of 400 to saturating level of 66000. 2 channelshybridization was performed and Cy3 signals were compared to Cy5signals, where fluor reversed chip was preformed (normal vs. disease),probe signal was set to be its average signal. Signals were normalizedby dividing them with the known miRNAs average signals such that the sumof known miRNAs signal is the same in each experiment or channel. Signalratios between disease and normal tissues were calculated. Signal ratiogreater than 1.5 indicates a significant upregulation with a P value of0.007 and signal ratio greater than 2 has P value of 0.003. P valueswere estimated based on the occurrences of such or greater signal ratiosover duplicated experiments.

The differential expression analysis in Table 2 indicates that theexpression of a number of the miRNAs are significantly altered indisease tissue. In particular, the MC19-1 miRNAs of Example 4 aredifferentially expressed in prostate and lung cancer. The relevance ofthe MC19-1 miRNAs to cancer is supported by the identification of a lossof heterozygosity within the MC19-1 region in prostate cancer derivedcells (Dumur et al. 2003).

1.-17. (canceled)
 18. An isolated nucleic acid of 18-200 nucleotides inlength comprising a sequence selected from the group consisting of: (a)SEQ ID NO: 550; (b) a DNA encoding the nucleic acid of (a), wherein theDNA is identical in length to (a); (c) a sequence at least 80% identicalto (a) or (b); and (d) the complement of (a), (b) or (c), wherein thecomplement is identical in length to the nucleic acid of (a), (b), or(c).
 19. An isolated nucleic acid of 18-100 nucleotides in lengthcomprising a sequence selected from the group consisting of: (a) SEQ IDNO: 8087 or a fragment thereof wherein the fragment is 18-22 nucleotidesin length; (b) a DNA encoding the nucleic acid of (a), wherein the DNAis identical in length to (a); (c) a sequence at least 80% identical to(a) or (b); and (d) the complement of (a), (b) or (c), wherein thecomplement is identical in length to the nucleic acid of (a), (b), or(c).
 20. A vector comprising a heterologous sequence, wherein theheterologous sequence consists of the sequence of the nucleic acid ofclaim
 18. 21. A vector comprising a heterologous sequence, wherein theheterologous sequence consists of the sequence of the nucleic acid ofclaim
 19. 22. A probe comprising a heterologous sequence, wherein theheterologous sequence consists of the sequence of the nucleic acid ofclaim
 18. 23. A probe comprising a heterologous sequence, wherein theheterologous sequence consists of the sequence of the nucleic acid ofclaim 19.